Interferon Alpha (IFNα, encoded by IFNA gene family) is a type I interferon with potent antiviral, antiproliferative, and immunomodulatory activities. It is primarily secreted by plasmacytoid dendritic cells, lymphocytes, and other host cells in response to viral infection, double-stranded RNA, and various pathogen-associated molecular patterns. Upon binding to its cell surface receptor complex IFNAR1/IFNAR2, IFNα initiates intracellular signaling cascades that induce the expression of hundreds of interferon-stimulated genes (ISGs), which inhibit viral replication, modulate immune cell function, and suppress tumor cell growth. Clinically, recombinant IFNα is widely used in the treatment of viral hepatitis, certain hematological malignancies, and some solid tumors. Dysregulation of IFNα signaling is linked to autoimmune diseases and chronic inflammatory disorders. IFNα binding triggers receptor dimerization and recruits TYK2, which mediates downstream JAK-STAT phosphorylation and signaling activation. Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse IFNα and recombinant human TYK2. Briefly, IFNα was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to TYK2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IFNα pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant mouse IFNα and recombinant human TYK2 was shown in Figure 1, the EC50 for this effect is 0.473 µg/mL.